Academic Dissertation to be presented with the assent of the Faculty of Medicine, University of Oulu, for public discussion in Auditorium 1 of the University Hospital of Oulu, on February 4th, 2000, at 12 noon.

Tarkastaja(t):

Docent Mikko Hippeläinen
Docent Matti Vapalahti

Kuvaus:

Abstract

The progress in recombinant DNA technology has made possible
the introduction of exogenous genetic material into cells. Gene
therapy aims to correct a defective gene, introduce a therapeutic exogenous
gene or a counteracting gene into somatic cells without modification
of the germ-cell line. The most important technical interests in
the field of gene therapy research have pertained to the development
of safe and effective vectors and suitable methods for the delivery
of the exogenous gene carrying vectors into the target cells. The
aim of this study was to evaluate surgical methods used for gene
delivery and to develop an effective gene transfer method for organ-specific
gene transfer, primarily into the renal glomeruli.

There are genetic and acquired diseases that are candidates
for gene therapy. Alport syndrome is an X-chromosome-linked disease
caused by a mutation in the type IV collagen α5 chain gene,
which causes a defect of the glomerular basement membrane in the
kidney, leading to progressive renal failure in males. This manifestation
could theoretically be prevented by the transfer of a normal α5 chain
gene into the renal glomerular cells. Cystic fibrosis and α1-antitrypsin
deficiency are examples of pulmonary diseases and genetic lysosomal
storage diseases that are candidates for splenic gene transfer.
The gene transfer strategies used so far have proved relatively
ineffective. Recombinant adenovirus, retrovirus, adeno-associated
virus and liposomes have been previously used as vectors. Direct
injection, intra-arterial, intravenous and intratracheal delivery
of vectors have been the most extensively studied methods.

This preclinical experimental work for marker gene transfer
into the kidney, spleen, lung and mammary gland was done by using
rabbits, pigs and goats as test animals. The adenoviral vector carrying
a β-galactosidase reporter gene was first infused in the
renal artery of rabbits and pigs in vivo with
or without pharmacological agents. This did not result in any remarkable
gene transfer into the kidney. Next, the incubation time between
the vector and the target cells was prolonged by ex vivo perfusion of explanted kidneys
for 12 hours. Perfusion at room temperature did not improve gene transfer.
When the perfusion temperature was raised to 37°C, improved
and mostly glomerular gene transfer was observed, with up to 80% of
the glomeruli showing β-galactosidase expression in four ex vivo experiments.

A closed-circuit organ perfusion method for in vivo gene transfer was developed
in this study. The surgical perfusion experiment was tested successfully
in ten in vivo perfusions of
the kidney, eight of the spleen and eight of the lung in a porcine
model. This method led to effective, up to 75% gene transfer
into the renal glomeruli as assessed after four days. In the spleen,
the perfusion method resulted in relatively effective gene transfer
into perifollicular splenic cells, mostly macrophages and endothelial
cells. Lung perfusion yielded transgene expression in alveolar epithelial
cells, bronchiolar epithelial cells and, to a lesser extent, arteriolar
endothelial cells and alveolar macrophages. Perfusion of the goats
mammary gland using a retroviral vector in three experiments resulted
in growth hormone secretion into the milk.

The gene transfer operation was well tolerated by the animals,
and no clinical signs of inflammation were observed. No remarkable
humoral immunological response against adenovirus or β-galactosidase
was elicited in the kidney experiments, but histological signs of
inflammation as mononuclear cell clusters in the kidney and lung
were seen four and seven days after the experiments. The spleen
showed no macroscopic or microscopic pathologic alterations after
the perfusion.